High-sensitivity array analysis of gene expression for the early detection of disseminated breast tumor cells in peripheral blood

Early detection is an effective means of reducing cancer mortality. Here, we describe a highly sensitive high-throughput screen that can identify panels of markers for the early detection of solid tumor cells disseminated in peripheral blood. The method is a two-step combination of differential display and high-sensitivity cDNA arrays. In a primary screen, differential display identified 170 candidate marker genes differentially expressed between breast tumor cells and normal breast epithelial cells. In a secondary screen, high-sensitivity arrays assessed expression levels of these genes in 48 blood samples, 22 from healthy volunteers and 26 from breast cancer patients. Cluster analysis identified a group of 12 genes that were elevated in the blood of cancer patients. Permutation analysis of individual genes defined five core genes (P ≤ 0.05, permax test). As a group, the 12 genes generally distinguished accurately between healthy volunteers and patients with breast cancer. Mean expression levels of the 12 genes were elevated in 77% (10 of 13) untreated invasive cancer patients, whereas cluster analysis correctly classified volunteers and patients (P = 0.0022, Fisher's exact test). Quantitative real-time PCR confirmed array results and indicated that the sensitivity of the assay (1:2 × 108 transcripts) was sufficient to detect disseminated solid tumor cells in blood. Expression-based blood assays developed with the screening approach described here have the potential to detect and classify solid tumor cells originating from virtually any primary site in the body.

Detection of synchrony in the activity of auditory nerve fibers by octopus cells of the mammalian cochlear nucleus

The anatomical and biophysical specializations of octopus cells allow them to detect the coincident firing of groups of auditory nerve fibers and to convey the precise timing of that coincidence to their targets. Octopus cells occupy a sharply defined region of the most caudal and dorsal part of the mammalian ventral cochlear nucleus. The dendrites of octopus cells cross the bundle of auditory nerve fibers just proximal to where the fibers leave the ventral and enter the dorsal cochlear nucleus, each octopus cell spanning about one-third of the tonotopic array. Octopus cells are excited by auditory nerve fibers through the activation of rapid, calcium-permeable, α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptors. Synaptic responses are shaped by the unusual biophysical characteristics of octopus cells. Octopus cells have very low input resistances (about 7 MΩ), and short time constants (about 200 μsec) as a consequence of the activation at rest of a hyperpolarization-activated mixed-cation conductance and a low-threshold, depolarization-activated potassium conductance. The low input resistance causes rapid synaptic currents to generate rapid and small synaptic potentials. Summation of small synaptic potentials from many fibers is required to bring an octopus cell to threshold. Not only does the low input resistance make individual excitatory postsynaptic potentials brief so that they must be generated within 1 msec to sum but also the voltage-sensitive conductances of octopus cells prevent firing if the activation of auditory nerve inputs is not sufficiently synchronous and depolarization is not sufficiently rapid. In vivo in cats, octopus cells can fire rapidly and respond with exceptionally well-timed action potentials to periodic, broadband sounds such as clicks. Thus both the anatomical specializations and the biophysical specializations make octopus cells detectors of the coincident firing of their auditory nerve fiber inputs.

Parallel detection of violations of color constancy

The perceived colors of reflecting surfaces generally remain stable despite changes in the spectrum of the illuminating light. This color constancy can be measured operationally by asking observers to distinguish illuminant changes on a scene from changes in the reflecting properties of the surfaces comprising it. It is shown here that during fast illuminant changes, simultaneous changes in spectral reflectance of one or more surfaces in an array of other surfaces can be readily detected almost independent of the numbers of surfaces, suggesting a preattentive, spatially parallel process. This process, which is perfect over a spatial window delimited by the anatomical fovea, may form an early input to a multistage analysis of surface color, providing the visual system with information about a rapidly changing world in advance of the generation of a more elaborate and stable perceptual representation.

Real-time detection of DNA hybridization and melting on oligonucleotide arrays by using optical wave guides.

The challenge of the Human Genome Project is to increase the rate of DNA sequence acquisition by two orders of magnitude to complete sequencing of the human genome by the year 2000. The present work describes a rapid detection method using a two-dimensional optical wave guide that allows measurement of real-time binding or melting of a light-scattering label on a DNA array. A particulate label on the target DNA acts as a light-scattering source when illuminated by the evanescent wave of the wave guide and only the label bound to the surface generates a signal. Imaging/visual examination of the scattered light permits interrogation of the entire array simultaneously. Hybridization specificity is equivalent to that obtained with a conventional system using autoradiography. Wave guide melting curves are consistent with those obtained in the liquid phase and single-base discrimination is facile. Dilution experiments showed an apparent lower limit of detection at 0.4 nM oligonucleotide. This performance is comparable to the best currently known fluorescence-based systems. In addition, wave guide detection allows manipulation of hybridization stringency during detection and thereby reduces DNA chip complexity. It is anticipated that this methodology will provide a powerful tool for diagnostic applications that require rapid cost-effective detection of variations from known sequences.